Chronic hepatitis C virus (HCV) infections leads to a high frequency of liver transplants in the U.S. as a result of complications of liver cirrhosis and long term virus persistence. The overall objective of this proposal is to investigate the function of the HCV-encoded nonstructural protein NS5A in virus- host interactions and in mediating resistance to type I interferon (IFN), which is the only approved therapy for these patients. Large majority of patients do not respond to or relapse after cessation of IFN therapy. Experimental evidence to date suggest that severity of infection and responsiveness to IFN therapy are subtype-related HCV-NS5A sequence variations. HCV- NS5A is a phosphoprotein and is thought to be a component of viral RNA replicase. But its precise role in the virus life cycle is unknown. Recently, it has been shown to associate with more than one cellular protein kinases. One of these kinases has been identified to be interferon (IFN)-inducible double-stranded RNA activated protein kinase (PKR) and the identity of other kinase(s) is unknown. A hypothesis is proposed in this application that the subtype variations in the intrinsic properties of HCV-NS5A to associate with PKR and other unidentified cellular kinase(s) modulate differentially the IFN signal transduction pathways and may contribute to resistance of HCV infections to IFN therapy. The overall objective of this proposal will be accomplished through the following Specific Aims: 1. To identify the cellular kinase(s) that associates with NS5A by using recombinant NS5A proteins expressed using E. coli and baculovirus expression systems. We will purify the kinase(s) by interaction affinity chromatographic methods and use a number of peptides as substrates which are previously characterized for well known protein kinases. We will carry out protein microsequence analysis of the purified kinase. 2. To dissect the molecular interactions of NS5A with the unidentified cellular kinase, PKR, and STAT1. A hepatocellular carcinoma (HepG2) cell line conditionally expressing NS5A under an inducible promoter will be isolated. The NS5A-associated kinase activities will be quantitatively correlated with the effect of IFN treatment of cells. We will investigate whether expression of NS5A interferes with IFN signaling pathways using immunoprecipitation analyses and electrophoretic mobility shift assays. 3. After completion of specific aims 1 and 2, we will identify the phosphorylation sites of NS5A utilized by the associated kinase in vitro as well as intracellularly as a result of IFN treatment by two- dimensional phosphopeptide mapping techniques and microsequence analyses.